In vitro PUVA treatment triggers calreticulin exposition and HMGB1 release by dying T lymphocytes in GVHD: New insights in extracorporeal photopheresis

Abstract

Background Extracorporeal photopheresis (ECP) is an effective therapy for graft vs host disease (GVHD), based on infusion of UVA‐irradiated and 8 methoxy‐psoralen (PUVA)‐treated leukocytes. Reinfusion of these apoptosing cells affects the functionality of pathogenic T cells through poorly understood immunomodulatory mechanisms. Apoptosis is usually a silent, tolerance‐associated process, but can also be immunogenic, depending on death‐inducers and environmental context. Methods To understand ECP mechanisms of action, human alloreactive T cells generated in an in vitro model mimicking GVHD were used, as well as primary cells from GVHD patients. Cells were submitted to PUVA treatment and their phenotype and immunogenicity were analyzed, using cell culture and flow cytometry. Results In vitro PUVA treatment induced the expression of several damage‐associated molecular patterns (DAMPs) by dying T cells (calreticulin, high‐mobility group box‐1, and to a lesser extent heat shock proteins 70 and 90), especially upon T cell activation, leading to their phagocytosis by macrophages and dendritic cells (DCs). Allogeneic DCs preincubated with PUVA treated T cells induced comparable naive T cell proliferation and polarization as control allogeneic DC. Conclusion Altogether, in our experimental settings, in vitro PUVA‐treatment induces a partially immunogenic phenotype allowing phagocytosis of apoptotic cells by macrophages and DC, however not sufficient to induce dendritic cell maturation and T cell activation. These data refine current models of ECP‐mediated immune modulation and emphasize the need to further analyze PUVA‐treated cell interactions with immune cells.

Does Offline Beat Inline Treatment: Investigation into Extracorporeal Photopheresis

“…Although extracorporeal photopheresis treatment is widely used as a second-line treatment of GvHD, the exact “pharmaceutical” mechanism of this therapy remains unclear. There are no hard facts or data points that can be used to predict the clinical efficacy of an ECP apheresis product [18, 19], although there is some indication that distinct cell populations within the recipient might be a prerequisite for successful treatment [20]. A factor that can be measured as quality control of ECP therapy is the dose of apoptotic MNCs [21] or the number of lymphocytes and MNCs/kg body weight given per single procedure [22].…”

High mobility group box 1 (HMGB1): a pivotal regulator of hematopoietic malignancies

“…Extracorporeal photopheresis (ECP) depends on infusion of UVA-irradiated and 8 methoxy-psoralen (PUVA)-treated leukocytes and is an effective treatment measure for GVHD. In vitro PUVA treatment induces the expression of HMGB1 in dying T cells, especially upon T cell activation, leading to their phagocytosis by macrophages and DCs [156]. In AML patients who received allo-HSCT, a higher γδ T cell count, which is an important early source of TNF-α and IFN-γ, predicted a better prognosis.…”

Human Apoptotic Cells, Generated by Extracorporeal Photopheresis, Modulate Allogeneic Immune Response

“…Although preclinical results are robust in animal models of transplantation, data on the characterization and immunosuppressive properties of apoptotic cells obtained from human samples remain rare. In vitro, it was suggested that 8-MOP-treated and UV-A-irradiated cells could acquire a partial immunogenic phenotype that triggers phagocytosis of apoptotic cells by macrophages and DC; however, it was not enough to induce DC maturation and T-cell activation (19). In the present study, we used apoptotic cells generated from human peripheral blood mononuclear cells (PBMCs) that were incubated with 8-MOP and exposed to UV-A radiation (referred to as Apo-cells); the procedure and the tools are clinically validated.…”